Abstractα-Conotoxins that are thought to act as antagonists of nicotinic acetylcholine receptors (nAChRs) containing α3-subunits are efficacious in several preclinical models of chronic pain. Potent interactions of Vc1.1 with other targets have suggested that the pain relieving actions of α-conotoxins might be mediated by either α9α10 nAChRs or a novel GABA B receptor-mediated inhibition of N-type calcium channels. Here we establish that three α-conotoxins, Vc1.1, AuIB and MII, have distinct selectivity profiles for these three potential targets. Their potencies after intramuscular administration were then determined for reversal of allodynia produced by partial nerve ligation in rats. Vc1.1, which potently inhibits α9α10 nAChRs and GABA B /Ca 2+ channels but weakly blocks α3β2 and α3β4 nAChRs, produced potent, long-lasting reversal of allodynia that were prevented by pretreatment with the GABA B receptor antagonist, SCH50911. α-Conotoxin AuIB, a weak α3β4 nAChR antagonist, inhibited GABA B /Ca 2+ channels but did not act on α9α10 nAChRs.AuIB also produced reversal of allodynia. These findings suggest that GABA B receptor-dependent inhibition of N-type Ca 2+ channels can mediate the sustained anti-allodynic actions of some α-conotoxins. However, MII, a potent α3β2 nAChR antagonist but inactive on α9α10 and α3β4 nAChRs and GABA B /Ca 2+ channels, was demonstrated to have short-acting anti-allodynic action.This suggests that α3β2 nAChRs may also contribute to reversal of allodynia. Together, these findings suggest that inhibition of α9α10 nAChR is neither necessary nor sufficient for relief of allodynia and establish that α-conotoxins selective for GABA B receptor dependent inhibition of Ntype Ca 2+ channels relieve allodynia, and could therefore be developed to manage chronic pain.

Abbreviations: Nav, voltage-gated sodium channel; TTX, tetrodotoxin; ProTxII, β/ω-theraphotoxin-Tp2a; Ca 2+ , calcium ion; DMEM, Dulbecco's Modified Eagle's Medium; FBS, foetal bovine serum; PBS, phosphate buffered saline; PSS, physiological salt solution; HBS, HEPES-buffered saline; AFU, arbitrary fluorescence unit; SEM, standard error of the mean; Fluo-4 AM, Fluo-4 acetoxymethylester; RPMI, Roswell Park Memorial Institute; BSA, bovine serum albumin; CCD, charge-coupled device; DAPI, 4',6-diamidino-2-phenylindole 2 AbstractThe human neuroblastoma cell line SH-SY5Y is a potentially useful model for the identification and characterisation of Nav modulators, but little is known about the pharmacology of their endogenously expressed Navs. The aim of this study was to determine the expression of endogenous Nav α and β subunits in SH-SY5Y cells using PCR and immunohistochemical approaches, and pharmacologically characterise the Nav isoforms endogenously expressed in this cell line using electrophysiological and fluorescence approaches. SH-SY5Y human neuroblastoma cells were found to endogenously express several Nav isoforms including Nav1.2 and Nav1.7. Activation of endogenously expressed Navs with veratridine or the scorpion toxin OD1 caused membrane depolarization and subsequent Ca 2+ influx through voltage-gated L-and N-type calcium channels, allowing Nav activation to be detected with membrane potential and fluorescent Ca 2 dyes. -Conotoxin TIIIA and ProTxII identified Nav1.2 and Nav1.7 as the major contributors of this response. The Nav1.7-selective scorpion toxin OD1 in combination with veratridine produced a Nav1.7-selective response, confirming that endogenously expressed human Nav1.7 in SH-SY5Y cells is functional and can be synergistically activated, providing a new assay format for ligand screening.Key Words: SH-SY5Y; Ca 2+ ; Nav1.7; ProTxII; OD1 3 IntroductionVoltage-gated sodium channels (Nav) are complex transmembrane proteins comprised of a poreforming α subunit and accessory β subunits that play an essential role in the initiation and propagation of action potentials in excitable cells. To date, apart from the related Nax which appears to function as a sodium sensor [1,2], nine isoforms termed Nav1.1 -Nav1.9 have been functionally defined as sodium-selective ion channels [3]. Their distinct tissue distribution and amenability to modulation by toxins and drugs has led to significant interest in Nav as therapeutic targets in a number of poorly treated conditions ranging from epilepsy to cardiac arrhythmias and pain [4]. In recent years Nav1.7 has emerged as an attractive drug target, with expression restricted to a subset of nociceptive neurons that is expected to limit on-target side effects of pharmacological modulators of Nav1.7 [5]. In addition, loss-of-function mutations in humans have highlighted the possibility that Nav1.7 inhibition could produce complete loss of pain sensations without dose-limiting side effects [6]. However, it remains unclear if such an effect can be translated to the clinic...

FXYD3, also known as mammary tumor protein 8, is overexpressed in several common cancers, including in many breast cancers. We examined if such overexpression might protect Na(+)/K(+)-ATPase and cancer cells against the high levels of oxidative stress characteristic of many tumors and often induced by cancer treatments. We measured FXYD3 expression, Na(+)/K(+)-ATPase activity and glutathionylation of the β1 subunit of Na(+)/K(+)-ATPase, a reversible oxidative modification that inhibits the ATPase, in MCF-7 and MDA-MB-468 cells. Expression of FXYD3 was suppressed by transfection with FXYD3 siRNA. A colorimetric end-point assay was used to estimate cell viability. Apoptosis was estimated by caspase 3/7 (DEVDase) activation using a Caspase fluorogenic substrate kit. Expression of FXYD3 in MCF-7 breast cancer cells was ~eightfold and ~twofold higher than in non-cancer MCF-10A cells and MDA-MB-468 cancer cells, respectively. A ~50 % reduction in FXYD3 expression increased glutathionylation of the β1 Na(+)/K(+)-ATPase subunit and reduced Na(+)/K(+)-ATPase activity by ~50 %, consistent with the role of FXYD3 to facilitate reversal of glutathionylation of the β1 subunit of Na(+)/K(+)-ATPase and glutathionylation-induced inhibition of Na(+)/K(+)-ATPase. Treatment of MCF-7 and MDA-MB- 468 cells with doxorubicin or γ-radiation decreased cell viability and induced apoptosis. The treatments upregulated FXYD3 expression in MCF-7 but not in MDA-MB-468 cells and suppression of FXYD3 in MCF-7 but not in MDA-MB-468 cells amplified effects of treatments on Na(+)/K(+)-ATPase activity and treatment-induced cell death and apoptosis. Overexpression of FXYD3 may be a marker of resistance to cancer treatments and a potentially important therapeutic target.

Voltage-gated sodium (NaV) channels are essential for the transmission of pain signals in humans making them prime targets for the development of new analgesics. Spider venoms are a rich source of peptide modulators useful to study ion channel structure and function. Here we describe β/δ-TRTX-Pre1a, a 35-residue tarantula peptide that selectively interacts with neuronal NaV channels inhibiting peak current of hNaV1.1, rNaV1.2, hNaV1.6, and hNaV1.7 while concurrently inhibiting fast inactivation of hNaV1.1 and rNaV1.3. The DII and DIV S3-S4 loops of NaV channel voltage sensors are important for the interaction of Pre1a with NaV channels but cannot account for its unique subtype selectivity. Through analysis of the binding regions we ascertained that the variability of the S1-S2 loops between NaV channels contributes substantially to the selectivity profile observed for Pre1a, particularly with regards to fast inactivation. A serine residue on the DIV S2 helix was found to be sufficient to explain Pre1a’s potent and selective inhibitory effect on the fast inactivation process of NaV1.1 and 1.3. This work highlights that interactions with both S1-S2 and S3-S4 of NaV channels may be necessary for functional modulation, and that targeting the diverse S1-S2 region within voltage-sensing domains provides an avenue to develop subtype selective tools.